Traditional methodologies for protein detection and quantification include 2-D gel electrophoresis, mass spectrometry and antibody binding. Each methodology has been used to quantify protein levels from relatively large amounts of tissue, yet each suffers from a lack of sensitivity.
The detection of low abundance antigens and their modifications is the key to understanding the function of many regulatory proteins that are critical for cellular events in the body as well as for the clinical diagnosis of infections and other pathological conditions. Limitations of immunologically detecting antigens relate to the concentration of the antigen and the affinity of the antibodies. Generally, monoclonal antibodies with higher affinity are able to detect antigens at lower concentrations. However the sensitivity of using antibody in detection is limited by detection of the bound antibody. Barriers to detection can be overcome if amplification of the binding signal of rare interactions could be generated in a linear and consistent manner.
The detection limit for ELISA type assays ranges from 0.1-50 ng/ml, depending upon the affinity of antibodies, especially the capture antibody used. In a “sandwich” or two-site ELISA assay, the use of higher affinity antibody to capture antigen correlates with higher sensitivities (Porstmann, T. & Kiessig, S. T. Enzyme immunoassay techniques. An overview. Journal of Immunological Methods. 150, 5-21 (1992)). The detection antibody is often conjugated to enzymes, usually peroxidase or alkaline phosphatase, to amplify the signals. Replacing calorimetric products with fluorogenic substrates only slightly improves the sensitivity of the assay by less than one order of magnitude (Porstmann, B., Porstmann, T., Nugel, E. & Evers, U. Which of the commonly used marker enzymes gives the best results in colorimetric and fluorimetric enzyme immunoassays: horseradish peroxidase, alkaline phosphatase or beta-galactosidase? Journal of Immunological Methods. 79, 27-37 (1985)). In addition, increased background is generally associated with improved sensitivity of ELISA assays using peroxidase or alkaline phosphatase as well as other non-linear enzymes.
Improvement of the ability to monitor proteins, lipids, sugars and metabolite levels and their modifications is needed for cell biology and medicine. A variety of technologies have been employed to improve the sensitivity of detecting these molecules. Recent examples of detection methods include immuno-PCR, RCA and immuno-aRNA.
Immuno-PCR (U.S. Pat. No. 5,665,539, which is incorporated herein by reference) combines the polymerase chain reaction (PCR) technology with conventional detection methods to increase the sensitivity to detect protein. However, a major limitation of immuno-PCR lies in the non-linear amplification ability of PCR reaction, which limits this technique as a quantitative detection method. Thus, this method provides no direct correlation between the amount of signal and the amount of protein present.
A relatively isothermal rolling circle DNA amplification technique (RCA; Schwietzer et al., Proc. Natl. Acad. Sci. USA 97, 10113, 2000), which is incorporated herein by reference) provides an improvement over immuno-PCR as this technique overcomes some of the quantitation problems associated with immuno-PCR. Rolling-circle amplification (RCA) has also been termed immunoRCA. The amplified signal (single strand DNA) stays with the antigen-antibody complex. ImmunoRCA is an attractive approach for protein microarrays. Femtomolar sensitivity was described for the immunoRCA approach but the actually detection sensitivity of cytokines studied with immunoRCA ranged from 1˜1000 pg/ml, a level comparable to already available ELISA detection.
Tannous et al (Nucleic Acids Research. 30, e140 (2002)) reported an antigen quantification system that used T7 RNA Polymerase. In their system, T7 RNA Polymerase was complexed with a detection antibody and a DNA template was supplied for amplification. Since the amplified RNA encoded either T7 RNA polymerase or luciferase, an in vitro translation system was then employed to produce enzymes and the final enzyme activity reflected the original antigen concentration.
U.S. Pat. No. 5,922,553, which is incorporated herein by reference, discloses a method for quantifying levels of a selected protein via a technique referred to as immuno-aRNA. In this method, a first antibody targeted to a selected protein is immobilized to a solid support. The support is then contacted with the selected protein so that the selected protein is immobilized to the first antibody. The solid support is then contacted with a RNA promoter-driven cDNA sequence covalently coupled to a second antibody targeted to the selected protein so that the second antibody binds to the bound selected protein. The amount of selected protein is determined by quantifying levels of the promoter driven cDNA sequence covalently coupled to the bound second antibody via an amplified RNA technique. In a preferred embodiment, a T7 promoter driven cDNA sequence is covalently coupled to the second antibody. Accordingly, the antigen of interest is captured by the plate-associated antibody and detected by antibodies directly coupled with double stranded oligonucleotide that accommodates the attachment of the T7 RNA polymerase enzyme. The interaction of T7 leads to the production of RNA species that is monitored with labeled nucleotide. The original concentrations of antigens are determined by auto-radiographic analyses of the RNA species after electrophoresis. While detection sensitivity is high due to the combination of T7 RNA polymerase amplification and use of radioactive isotopes, it also had significant drawbacks. Labeling the amplification products (RNA) with radioisotope and separation of the RNA species with electrophoresis creates a set of lengthy and often difficult steps that preclude widespread usage. Covalent glutaraldehyde coupling conditions are intrinsically variable and have an unpredictable effect on antibody affinity and function.
Single chain fragments as well as exocyclic peptide based complementarity determining region (CDR) subunits have been found to be useful in this immuno-aRNA technique. Further, it has been found that PCR, as well as amplified RNA techniques, can be used to quantify the promoter driven cDNA sequence covalently coupled to the bound single chain fragment or CDR subunit. The use of smaller antibody binding units and fragments coupled with the already existing large single chain or cyclic peptide libraries and the use of robotic assistance renders this method widely useful for both medicinal and research purposes. Furthermore, a single third detector species can be coupled with double-stranded DNA and bound to either the single chain Fv or the CDRs, rendering detection uniform and simple.